科研之友
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摘要
In ventricular myocytes, alpha 1-AR stimulates G alpha s proteins and reduces the transient outward K+ current (I-to) via a caMp/PKA-mediated pathway and thus regulates cardiac contraction and excitability. This I-to reduction is compartmentalized and limited to discrete membrane regions since PKA-dependent phosphorylation of the I-to channels after alpha 1-AR stimulation requires the integrity of both the sarcoplasmic membrane and the cytoskeleton. The aim of this work was to investigate the mechanisms involved in the compartmentalization of the PKA-dependent modulation of I-to in response to alpha 1-AR activation. I-to current recordings were performed by the patch-Clamp technique. Membrane rafts from isolated ventricular myocytes were extracted by centrifugation in a sucrose density gradient. The different proteins were visualized by western blot and protein-protein interactions determined by coimmunoprecipitation experiments. Localization of I-to channel in caveolae, particular subtypes of membrane rafts, was achieved by electron microscopy. Patch-Clamp recordings show that a functional supramolecular complex, kept together by the a kinase anchoring protein AKAP100, exist in caveolae in living myocytes. Density gradients and immunoprecipitation experiments show that the components of the alpha 1-AR/I-to pathway localize in caveolae, forming two different groups of proteins. The K(V)4.2/K(V)4.3 channel forms a supramolecular complex with PKA through AKAP100 and is attached to caveolae by interacting with caveolin-3. On the other hand, alpha 1-AR, G alpha s and adenylate cyclase gather in a second group also connected to caveolin-3. Therefore, both groups of preassembled proteins are maintained in close proximity by caveolin-3. A different I-to channel population localizes in non-caveolar membrane rafts and is not sensitive to alpha 1-adrenergic regulation.
- 出版日期2010-6
